Transcriptome analysis of the role of SMAD6 in mediating endothelial cell response to laminar shear stress by high throughput RNA sequencing. Transcriptome analysis of the role of SMAD6 in mediating endothelial cell response to laminar shear stress by high throughput RNA sequencing

Laminar shear stress regulates blood vessel morphogenesis and subsequent quiescence, but how endothelial cells (EC) enact and maintain the vascular homeostasis required in most vessels for proper vessel function is poorly understood. SMAD6, a scaffold for several signaling pathways, is expressed in developing arteries and its expression is flow-regulated. We found that SMAD6 is essential for endothelial cell flow-mediated responses, and that it functions downstream of the mechanosensor Notch1. Endothelial cells with reduced SMAD6 levels failed to align under stable laminar shear flow that promotes vascular homeostasis, while forced SMAD6 expression rescued misalignment induced by reduced Notch1 signaling. SMAD6-dependent homeostatic laminar flow required the Notch ligand Dll4 and Notch transcriptional activity. Mechanistically, neither the N-terminal nor the C-terminal domain of SMAD6 alone rescued flow alignment upon loss of Notch signaling. Endothelial cells with reduced Smad6 levels has compromised barrier function, and RNA profiling revealed upregulation of proliferation-associated genes and down regulation of junction-associated genes. Among junction-related genes affected by SMAD6 levels, the proto-cadherin PCDH12 was upregulated by homeostatic flow and required for proper flow-mediated endothelial cell alignment. Thus, SMAD6 is a critical integrator of flow-mediated signaling inputs downstream of Notch1, as vessels transition from an angiogenic to a homeostatic phenotype. Overall design: To understand how SMAD6 mediate EC flow response, we performed bulk RNAseq of human umbilical vein endothelial cells (HUVEC) transfected with siNT non-targeting control siRNA or siSMAD6 (30 hrs of transfection) and then cultured under static condition or flowed under 15 dynes/cm2 laminar shear for 72 hrs.

层流剪切应力可调控血管形态发生及后续的血管静息状态，但内皮细胞（Endothelial Cells, EC）如何实现并维持大多数血管正常功能所需的血管稳态，目前尚不清楚。SMAD6作为多种信号通路的支架蛋白，在发育中的动脉中表达，且其表达受血流调控。我们发现SMAD6对于内皮细胞的血流介导应答至关重要，且其作用于机械感受器Notch1的下游。SMAD6水平降低的内皮细胞无法在促进血管稳态的稳定层流剪切环境下发生定向排列，而强制过表达SMAD6可挽救因Notch1信号减弱诱导的排列紊乱。SMAD6依赖的稳态层流过程需要Notch配体Dll4以及Notch的转录活性。机制层面，单独的SMAD6 N端结构域或C端结构域均无法挽救Notch信号缺失时的血流排列缺陷。SMAD6水平降低的内皮细胞屏障功能受损，转录组分析显示增殖相关基因上调、连接相关基因下调。在受SMAD6水平影响的连接相关基因中，原钙粘蛋白PCDH12可被稳态血流上调，且对于血流介导的内皮细胞正常排列必不可少。综上，当血管从血管生成表型转变为稳态表型时，SMAD6是Notch1下游血流介导信号输入的关键整合因子。实验整体设计：为阐明SMAD6如何介导内皮细胞的血流应答，我们对转染了非靶向对照小干扰RNA（siNT）或靶向SMAD6的小干扰RNA（siSMAD6）的人脐静脉内皮细胞（human umbilical vein endothelial cells, HUVEC）进行批量RNA测序：细胞转染30小时后，分别置于静态培养条件，或15达因/平方厘米的层流剪切环境中培养72小时。